Construction and transfection efficiency analysis of 5/11 chimeric adenovirus harboring RGD-4C
10.3724/SP.J.1008.2010.00178
- Author:
Hui LIU
1
Author Information
1. XinYuan Institute of Medicine and Biotechnology
- Publication Type:Journal Article
- Keywords:
Adenovirus vectors;
Hepatocarcinoma;
HI loop;
RGD-4C
- From:
Academic Journal of Second Military Medical University
2010;31(2):178-182
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To construct a 5/11 chimeric adenovirus harboring RGD-4C,and observe its transfection efficiency after transfecting it into hepatocarcinoma cell lines HepG2, BEL-7404 and BJ cells. Methods: RGD-4C was inserted into the HI loop region of 5/11 chimeric adenovirus fiber gene and the insertion outcome was confirmed by enzyme digestion and PCR analysis. The confirmed plasmid was co-transfected into E. coli BJ5183 together with adenoviral backbone plasmid pPE3 to produce recombinant plasmid by homologous recombination. Recombinants were selected and co-transfected into 293 cell line with PDC328-EF1-EGFP to produce recombinant adenovirus. The recombinant adenovirus production was confirmed by PCR analysis and was amplified and purified. The virus titer of recombinant adenovirus was determined. AD11-RGD-4C-EGFP was used to infect HepG2, BEL-7404, and BJ cell lines, and their transduction efficiency was determined by fluorescence microscope. Results: A 1 123 bp target gene fragments was obtained by PCR from the recombinant adenovirus; meanwhile, we prepared high titer recombinant adenovirus, with the titer being 1 3X10 10pfu/ml after purification. At 10 MOI, the infection efficiency of recombinant adenovirus was much higher than control adenovirus(AD11-EGFP) after 48 h infection. Conclusion: We have successfully constructed a chimeric adenovirus harboring RGD-4C, which paves a way for enhancing the infection efficiency of adenovirus in bio-therapy.
